lncRNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 Promotes Proliferation and Invasion of Non-Small Cell Lung Cancer Cells via Down-Regulating miR-202 Expression

Objective Accumulating evidences indicate that long non-coding RNAs (lncRNAs) play key roles in cancer. This study aims to clarify role of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in non-small cell lung cancer (NSCLC) and uncover the underlying mechanisms. Materials and Methods In this experimental study, MALAT1 and miR-202 expression in tissues and cell lines were detected using quantitative real time polymerase chain reaction (qRT-PCR) assay. Cell transfection was conducted using Lipofectamine 3000. Cell proliferation was determined with CCK-8 assay. MMP2 and MMP9 expressions were measured with Western blot. Cell invasive ability was evaluated by Transwell assay. Starbase 2.0 tool was used to predict targets of MALAT1. Dual luciferase reporter assay, RNA-binding protein immunoprecipitation assay and RNA pull-down assay were conducted to confirm the potential direct interaction between MALAT1 and miR-202. Results MALAT1 was overexpressed in NSCLC samples and cell lines. High expression of MALAT1 was related to large tumor size (>3 cm), poor histological grade, advanced cancer and tumor metastasis in NSCLC. In vitro assays exhibited that knockdown of MALAT1 remarkably decreased A549 cell growth and invasion capacity, while overexpression of MALAT1 significantly enhanced NCI-H292 cell proliferation and invasion ability. Next, we verified that MALAT1 could act as a competing endogenous RNA (ceRNA) by sponging miR-202 in NSCLC and there is a negative correlation between MALAT1 and miR-202. Besides, overexpression of miR-202 inhibited cell proliferation and invasive ability in MALAT1-overexpressed cells. Conclusion This study demonstrated that lncRNA-MALAT1 gets involved in NSCLC progression by targeting miR- 202, indicating that MALAT1 may serve as a novel therapeutic target for NSCLC treatment.


Introduction
Lung cancer is the first cause of cancer deaths worldwide, leading to about 1.6 million patients die per year (1). According to the pathological diagnosis, lung cancer is divided into small-cell lung cancer (SCLC, around 15%) and non-small cell lung cancer (NSCLC, around 85%). Although diagnostic techniques and therapy strategies (such as surgical techniques and targeted treatment) have progressed, the 5-year overall survival rate is still below 15%. Besides, this 15% of patients are accompanied with high recurrence rates (2). Thus, it is necessary to determine oncogenes involved in lung cancer development and progression and explore the underlying mechanism, facilitating development of more effective treatment methods.
Long noncoding RNAs (lncRNAs) are an emerging class of transcripts, which is longer than 200 nucleotides (nt). Although lncRNAs are coded by the genome, they are hardly translated into proteins. Previous researches revealed that lncRNAs serve as new regulators, controlling gene expressions epigenetically and post-transcriptionally. They also play crucial roles in modulating chromatin dynamics, cell growth, differentiation and development (3). Increasing evidences indicated that many lncRNAs are observed to be abnormally expressed in many types of cancer (4). For instance, Wei and Wang (5) found that lncRNA-MEG3 was downregulated in gastric carcinoma specimens and overexpression of it could repress gastric cancer cell growth and mobility via elevating p53 expression. lncRNA CPS1-IT1 was reported to serve as tumor suppressor in colorectal cancer and low CPS1-IT1 expression indicated poor prognosis (6).
The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also called as nuclear-enriched abundant transcript 2 (NEAT2), HCN, LINC00047, NCRN00047 and PRO2853, is an extensively expressed lncRNA, with the length of around 8000 nt (7). In 2003, MALAT1 was first found to function as a survival LncRNA MALAT1 Promotes NSCLC Progression via Targeting miR-202 prognostic factor for stage I lung adenocarcinoma or squamous cell carcinoma patients (8). In recent years, accumulating evidences suggested that MALAT1 plays a key role in tumorigenesis. In gastric cancer, MALAT1 was reported to promote tumorigenicity and metastasis through facilitating vasculogenic mimicry and angiogenesis (9). In triple-negative breast cancer, MALAT1 was found to promote cell proliferation and invasion via decreasing expression of miR-129-5p (10). Xie et al. (11) revealed that MALAT1 suppressed apoptosis and enhanced cell invasion ability via inhibiting miR-125p in bladder cancer. In epithelial ovarian cancer, MALAT1 was found to facilitate cell growth and induce epithelial-mesenchymal transition (EMT) through modulating PI3K/AKT signaling pathway (12). The study performed by Li et al. (13) showed that MALAT1 is positively correlated with chemoresistance in colorectal cancer patients. Nevertheless, further investigations are still required to identify role and function of MALAT1 in development and progression of NSCLC.
Previous studies have identified miR-202 as a tumor suppressor. For instance, in papillary thyroid carcinoma, miR-202 attenuates cell migration and invasion abilities via inhibiting Wnt signaling pathway (14). In human bladder cancer, miR-202 suppresses cell growth and metastasis through targeting EGFR (15). Furthermore, miR-202 was found to reduce expression level of TGFβ receptors and reverse TGFβ1-mediated EMT in pancreatic cancer (16). In NSCLC, miR-202 decreased cell viability and weakens cell mobility and invasive capacity by suppressing STAT3 activity (17).
In this study, we observed that lncRNA-MALAT1 was highly expressed in NSCLC tissues and cell lines. Correlation analysis revealed that high MALAT1 expression was related to large tumor size (> 3 cm), moderate or poor differentiation, advanced tumor stage and metastasis. Biologically functional experiments demonstrated that MALAT1 promoted NSCLC cell proliferation and invasion. Further molecular mechanisms revealed that MALAT1 could sponge miR-202 within NSCLC progression.

Patients and tissue samples
Forthy NSCLC tissues as well as corresponding adjacent normal tissues specimens were collected from Guangzhou Panyu Hospital of Chinese Medicine between June 2015 and July 2018. Patients involved in this study had not received any preoperative radiotherapy or chemotherapy. All specimens were identified as NSCLC tissues or normal lung tissues via histopathological observation. After resection, all tissues were dipped in liquid nitrogen promptly and then were stored at -80˚C for further studies. All enrolled patients were informed to sign the written informed consent and this study was approved by the Ethics Committees of Guangzhou Panyu Hospital of Chinese Medicine (license number of ethics statement: 2015HW126).

RNA extraction and quantitative real time polymerase chain reaction assay
Total RNA was extracted from tissue specimens and cell lines by using TRIzol reagent (Invitrogen, USA) according to manufacturer's protocol and treated with DNase I (Thermo Fisher Scientific, USA) to remove genomic DNA. cDNA was synthesized with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland). For miRNAs, reverse transcription was conducted with TaqMan Micro-RNA Reverse Transcription Kit (Applied Biosystems, USA). Expression level of lncRNA-MALAT1 was analyzed on a CFX96 real-time thermocycler (BioRad, USA) by using SsoAdvanced™ Universal SYBR ® Green Supermix (BioRad, USA). Detection of miR-202 was performed using TaqMan microRNA Assay kit (Applied Biosystems, USA) on the CFX96 real-time thermocycler (BioRad, USA). GAPDH and U6 were considered as endogenous control of lncRNA-MALAT1 and miR-202 respectively. Relative expression levels were calculated by using 2 -ΔΔCT method. All primers used in this study are listed below: The sequences of MALAT1 were quoted from Zuo et al. (10). The sequences of miR-202 were quoted from Hoffman et al. (18), while the sequences of GAPDH and U6 were designed by ourselves using Pubmed. scramble oligonucleotides and pcDNA3.1-MALAT1 were supplied by GenePharma company (Shanghai, China). Transfection was conducted with Lipofectamine 3000 Reagent (Thermo Fisher Scientific, USA) in accordance with the manufacturer's instruction. The sequence of siRNAs against MALAT1 were as follows: si-MALAT1: 5′-GAGCAAAGGAAGUGGCUUA-3′ si-NC: 5′-CGUACGCGGAAUA CUUCGAdTdT-3′.

Western blot assay
Total protein was extracted from cell pellet using RIPA lysis buffer (Thermo Fisher Scientific, USA) supplemented with protease inhibitors and phosphatase inhibitors (Roche) according to the manufacturer's protocol. Concentration of total protein was determined by using BCA™ Protein Assay Kit (Thermo Fisher Scientific, USA). Then, 40 μg of protein per lane was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. 5% skim milk was used to block PVDF membranes for 1 hour at room temperature. Next, membranes were incubated with primary antibodies overnight at 4˚C, followed by incubation of secondary antibodies for 1 hour at room temperature. Next, protein bands were visualized using the enhanced chemiluminescence system (Bio-Rad Clarity Western ECL, USA). Primary antibodies, including MMP2 (1:1000), MMP9 (1:1000) and β-actin (1:1000) and HRP-conjugated secondary antibodies (1:5000) were obtained from Cell Signaling Technology (CST Inc., USA). β-actin was regarded as the internal control.

Transwell invasion assay
24-well Transwell chambers were purchased from Corning (USA). After 24 hours transfection, 1×10 4 suspended cells in 100 μl serum free medium were seeded in upper chambers smeared on Matrigel (BD Biosciences, USA). Bottom chambers were filled with 600 μl medium containing 10% FBS. After 48 hours culture, upper chambers were fixed with 4% formaldehyde and stained with 0.05% crystal violet. Then, a cotton swab was used to rub away cells on the above membrane. The invaded cells through membrane were counted using optical microscopy.

Dual-luciferase reporter gene assay
Firstly, the full-length 3′-UTR of MALAT1 with miR-202 binding sites was cloned into the downstream of firefly luciferase gene in pGL3 (Invitrogen, USA) to construct pGL3-MALAT1 wild type (WT) and mutant (Mut). HEK293T cells were co-transfected with WT-MALAT1, Mut-MALAT1 reporter gene plasmid or pRL-TK plasmids and miR-202 mimics or miR-NC with Lipofectamine 3000 (Thermo Fisher Scientific, USA). The pRL-TK Vector was intended for use as an internal control reporter vector and may be used in combination with any experimental reporter vector to co-transfect mammalian cells. The pRL-TK Vector contains the herpes simplex virus thymidine kinase (HSV-TK) promoter to provide low to moderate levels of Renilla luciferase expression in co-transfected mammalian cells. 48 hours later, luciferase activity was determined with a dual-luciferase reporter assay system (Promega, USA).

RNA-binding protein immunoprecipitation assay
A Magna RIP RNA binding protein immunoprecipitation kit was obtained from Millipore (Darmstadt, German) and the Ago2 antibody was purchased from Abcam (Cambridge, USA). RIP assay was conducted using the magna RIP RNA binding protein immunoprecipitation kit and Ago2 antibody in accordance with the instruction of manufacturer. qRT-PCR was used to determine expression level of co-precipitated RNAs.

RNA pull-down assay
Biotin-labeled miR-NC and biotin-labeled miR-202 were synthesized by GenePharma company (Shanghai, China). 48 hours after transfection with biotin-labeled miR-NC or biotin-labeled miR-202, the cells were collected to conduct an RNA pull-down experiment using Pierce TM Magnetic RNA Protein Pull-down Kit (Thermo Fisher Scientific, USA) following the manufacturer's instruction. lncRNA-MALAT1 level was determined using qRT-PCR from the pull-down samples.

Statistical analysis
Statistical analyses were processed with GraphPad Prism 6.0 software (GraphPad software, USA) and all data were expressed as mean ± standard deviation (SD). Student t test or one-way ANOVA was used to determine the differences between two groups or among multiple groups respectively. P<0.05 was considered as statistically significant.

Overexpression of lncRNA-MALAT1 is observed in NSCLC tissues and cell lines
To investigate the role of lncRNA-MALAT1 in development of NSCLC carcinogenesis, we analyzed lncRNA-MALAT1 expression in 40 paired NSCLC tissues and pericarcinomatous normal tissues with qRT-PCR. As shown in Figure 1A, the expression level of lncRNA-MALAT1 was notably higher in NSCLC tissue samples than that in pericarcinomatous normal tissue (P<0.05). To further analyze the relationship between lncRNA-MALAT1 expression and clinical pathological parameters, 40 NSCLC patient samples were classified into two groups in accordance with the median relative quantity of lncRNA-MALAT1. The lncRNA-MALAT1 expression LncRNA MALAT1 Promotes NSCLC Progression via Targeting miR-202 levels above the median expression were defined as high expression while low expression of lncRNA-MALAT1 was termed as the expression was below the median level. Results showed that high lncRNA-MALAT1 expression significantly associate with tumor size (>3 cm), moderate or poor differentiation carcinoma, advanced tumor stage (namely advanced TNM stage, including III and IV stages) and tumor metastases (Fig.1B-E, P<0.05). In addition, we confirmed the expression level of lncRNA-MALAT1 in NSCLC cell lines. As shown in Figure 1F, up-regulation of lncRNA-MALAT1 was observed in NSCLC cell lines (A549, NCI-H23, NCI-H292, NCI-H1299 and NCI-H1975) compared to the normal lung cell BEAS-2B, indicating that lncRNA-MALAT1 may play a promotor role in NSCLC. Furthermore, A549 cell expressed the highest level of lncRNA-MALAT1 and NCI-H292 cell expressed the lowest level of lncRNA-MALAT1, compared to the other cell lines. Hence, A549 cell was chosen for silencing lncRNA-MALAT1 and overexpression of lncRNA-MALAT1 was performed on NCI-H292 cell.

lncRNA-MALAT1 binds to miR-202 and reduces its expression
As there is complementary sequence of miRNA in lncRNA, they can act as a competing endogenous RNAs, regulating miRNA expressions and biological function (19). To explore the mechanism of lncRNA-MALAT1 in progressing NSCLC, we used bioinformatics analysis web, Starbase 2.0 (http://starbase.sysu.edu.cn), to predict targets of lncRNA-MALAT1. We found that lncRNA-MALAT1 has a potential binding site to miR-202 (Fig.4A). We next performed dual luciferase reporter gene assay to confirm if miR-202 binds to the 3´-UTR of lncRNA-MALAT1 directly. Luciferase activity was markedly attenuated in HEK293 T cells co-transfected with MALAT1-WT plasmids and miR-202 mimics (P<0.05), while there was no change in the cells co-transfected with MALAT1-Mut plasmids and miR-202 mimics (Fig.4B). This indicates that the 3´-UTR of lncRNA-MALAT1 complementarily pairs to miR-202. Moreover, RIP assay showed that lncRNA-MALAT1 and miR-202 were both enriched in the Ago2 pellet compared to the IgG group (Fig.4C, P<0.05). Additionally, RNA pull-down assay presented that endogenous MALAT1 was pulled-down specifically in the cells overexpressing miR202 compared to the NC group (Fig.4D, P<0.05). This data suggested that miR-202 is a suppressive target of lncRNA-MALAT1.

lncRNA-MALAT1 promotes NSCLC cells proliferation and invasion via decreasing miR-202
Next, we conducted rescue experiments via overexpressing miR-202 in MALAT1-overexpressed cells to investigate whether or not miR-202 gets involved in MALAT1-mediated carcinogenesis. CCK-8 assay revealed that proliferation rate of the cells cotransfected with MALAT1 and miR-202 mimics was significantly reduced compared to that of MALAT1overexpressed cells (Fig.6A, P<0.05). In addition, ectopic expression of miR-202 down-regulated expression levels of MMP2 and MMP9 in MALAT1overexpressed cells, compared to MALAT1 group (Fig.6B, C, P<0.05). In addition, Transwell invasion assay presented that miR-202 mimics attenuated cell invasion capacity on MALAT1-overexpressed cells (Fig.6D, E, P<0.05). All together, these data demonstrated that lncRNA-MALAT1 promoted NSCLC cell proliferation and invasion partially by inhibiting miR-202 expression.

Discussion
More and more evidences have shown that the aberrant expression of lncRNAs was observed in lung cancer tissues, indicating that lncRNAs play multiple roles in carcinogenesis of lung cancer (20). For instance, Nie et al. (21) found that lncRNA urothelial carcinoma-associated 1 (UCA1) served as an oncogene in NSCLC. High expression of lncRNA-UCA1 predicted short survival time and multivariate analysis indicated that UCA1 was an independent risk parameter of prognosis. Chen et al. (22) reported that upregulation of small nucleolar RNA host gene 20 (SNHG20) was notably correlated with advanced tumor stage, lymph node metastases and larger tumor size, as well as poorer overall survival chance. Wang et al. (23) revealed that lncRNA-XIST contributed to cell proliferation and invasion by inhibition of miR-186-5p in NSCLC. Biological function of lncRNA-HIT (HOXA transcript induced by TGFβ) has been investigated in NSCLC. Results demonstrated that lncRNA-HIT facilitated NSCLC cell growth through interacting with E2F1 to regulate its target genes (24). Meanwhile, some lncRNAs conferring suppressive function in NSCLC have been recognized. For example, TUG1 (taurineupregulated gene 1) functions as a tumor suppressor in NSCLC (25). Considering their roles in tumorigenesis, lncRNAs may hold promise as diagnostic or prognostic biomarkers for lung cancer.
MALAT1 can be used to predict survival chance of stage I lung cancer or squamous cell cancer patients and it is phase and histologically specific to the metastasis of NSCLC patients (8). Accumulating studies have revealed that MALAT1 not only plays a pivotal role in NSCLC progression, but also promotes other kinds of tumors. Zhang et al. found that serum exosome-derived lncRNA-MALAT1 facilitated the tumor growth and migration, while it reduced apoptosis rate in NSCLC (26). The study performed by Li et al. (27) showed that MALAT1 facilitated NSCLC cell growth, colony formation and apoptosis by targeting miR-124. In ovarian cancer, MALAT1 was reported to facilitate cell proliferation and metastasis. It also prevents tumor cells from apoptosis (12). In this study, we found that MALAT1 was overexpressed in NSCLC tissues and cell lines (A549, NCI-H23, NCI-H292, NCI-H1299 and NCI-H1975) compared to corresponding adjacent normal tissues and normal lung cell BEAS-2B, respectively. This finding was in accordance with previous studies. Further correlation analysis demonstrated that high MALAT1 expression was positively related to large tumor size, poor histological grade, terminal stage of cancer and tumor metastasis. Knockdown of MALAT1 inhibited A549 cell growth and invasion, as well as the expression of MMP2 and MMP9. In contrary, overexpression of MALAT1 elevated NCI-H292 cell proliferation, invasion ability as well as the expression level of MMP2 and MMP9. These data indicated that MALAT1 functions as oncogene in NSCLC, which is in line with previous studies.
The ceRNA theory proposes that lncRNAs sharing miRNA response elements (MREs) with mRNAs can act as miRNA decoys. It has been reported that lncRNAs can act as ceRNA by sponging miRNAs in cancer progression (28). The underlying molecular mechanisms involved in lncRNAs interacting with miRNAs are as follows: i. lncRNA indirectly inhibits negative regulation of miRNAs on target genes by competing with miRNAs to bind to the 3´-UTR of target gene mRNA, ii. Some lncRNAs form miRNA precursors by intracellular cleavage, which is then processed into specific miRNAs, regulating expression of the target genes, iii. Some lncRNAs function as endogenous miRNA sponges inhibiting miRNA expression (29). For instance, in gastric cancer, lncRNA-HOTAIR was reported to serve as a ceRNA to modulate HER2 expression via sponging miR-331-3p (30). Huang et al. (31) found that lncRNA-CASC2 could function as a ceRNA through sponging miR-18a in colorectal cancer. In the present study, targets of MALAT1 were predicted by Starbase 2.0 (http://starbase.sysu.edu.cn). Then we used dualluciferase reporter gene assay, RIP assay as well as RNA pull-down assay to confirm that miR-202 was a direct target of MALAT1. MiR-202, a new tumor suppressor, is down-regulated in gastric cancer (32). In addition, miR-202 inhibits cell growth and promotes apoptosis in osteosarcoma through decreasing expression of Gli2 (33). miR-202 also restrains cell proliferation in human hepatocellular cancer via suppressing LRP6 expression post-transcriptionally (34). In prostate cancer, miR-202 inhibits cell proliferation and metastasis by inhibiting PIK3CA (35). Sun et al. (36) found that miR-202 can increase therapeutic effect of cisplatin against NSCLC via inhibiting activity of the Ras/MAPK pathway. Zhao et al. (37) revealed that up-regulation of miR-202 significantly reduces NSCLC cell viability, migration and invasion, and they suggested that STAT3 should be a direct target of miR-202. In this study, downregulation of miR-202 was observed in NSCLC compared to normal tissues. In addition, there was a negative correlation between MALAT1 and miR-202 expression in NSCLC tissues. Overexpression of miR-202 could reverse oncogenic effect of MALAT1 in NSCLC, indicating that miR-202 plays a key role in MALAT1-induced cell proliferation and metastasis in NSCLC cells.
The molecular mechanism whereby MALAT1 contributes to cancer progression appears to be diverse in different cancers. In gastric cancer, MALAT1 was found to increase cell viability via modulating SF2/ASF (38). In esophageal squamous cell carcinoma, MALAT1 promotes cell growth and invasion via regulating ATM-CHK2 signaling (39). In colorectal cancer, MALAT1 facilitates cell growth, mobility and invasion through targeting PRKA kinase anchor protein 9 (AKAP-9) (40). In ovarian cancer, MALAT1 contributes to cell EMT through modifying PI3K/AKT signaling pathway (12). In the present study, MALAT1 enhances cell proliferation and metastasis by sponging miR-202 in NSCLC cell lines. To our knowledge, this is the first report revealing interaction of MALAT1 with miR-202 in NSCLC. However, the molecular mechanism of miR-202 downregulation through MALAT1 activity requires further study.

Conclusion
This study elucidated that MALAT1 could facilitate cell growth and invasion via sponging miR-202 in NSCLC. Thus, our research demonstrated a new axis of MALAT1/ miR-202, suggesting a feasible therapeutic means for NSCLC treatment.